Diacylglycerol is a central mediator of downstream signaling by a host of hormones coupled through Gq and phospholipase Cbeta, growth factors coupled to tyrosine-kinase linked receptors and phospholipase Cgamma, and many other extra- and intracellular stimuli. The protein kinase C (PKC) isozyme family has historically been the most intensively studied class of targets for diacylglycerol signaling. PKC isoforms are under active investigation as therapeutic targets for cancer and retinopathy in diabetes, and as targets for immunosuppression. In response to diacylglycerol, conventional and novel PKC isozymes, and the protein kinase D isozymes, translocate to membranes, where they phosphorylate Ser and Thr residues in diverse proteins. The diacylglycerol-responsive PKC isozymes are also activated by phorbol esters, which acts as a potent agonist by binding to the same site as diacylglycerol. These PKC isozymes are activated by diacylglycerol and phorbol esters by virtue of their direct binding to motifs known as protein kinase C homology-1 (C1) domains. [unreadable] C1 domains are compact zinc fingers of 50-51 amino acids. Their structure comprises two small beta sheets and a single helix folded around two zinc ions. The two strands in the smaller beta sheet are pulled apart, due to a break in their hydrogen bonding induced by a conserved Pro residue. Phorbol ester binds stereospecifically in the groove formed where the two strands pull away from each other, first described by structural studies from this laboratory. The stereospecific phorbol ester binding site is surrounded on three side by bulky hydrophobic side chains, which form a hydrophobic wall around the phorbol ester binding site. Distal to the phorbol ester-binding site, Arg, Lys, and His residues form a basic belt on the surface of the C1 domain. The hydrophobic wall inserts in the hydrocarbon core of phospholipid bilayers upon binding, while the basic belt interacts with acidic phospholipid headgroup. The high affinity of C1 domainphorbol ester interactions in the presence of bilayers or micelles is due to the synergism between the stereospecific phorbol ester binding and nonspecific binding of acidic phospholipids to the basic and hydrophobic exterior surface of the C1 domain. Translocation and activation of C1 domain proteins requires the interplay of specific and nonspecific activators: both diacylglycerol or phorbol ester and bulk phospholipid.[unreadable] In recent years, it has become clear that there are several major classes of C1 domain-containing diacylglycerol receptors in addition to PKC. Munc-13 proteins are phorbol ester-binding scaffolding proteins involved in calcium-stimulated exocytosis. RasGRPs are diacylglycerol-activated guanine-nucleotide exchange factors (GEFs) for Ras and Rap1. The alpha- and beta-chimaerins are a family of phorbol ester- and diacylglycerol-responsive GTPase activating proteins. This project takes a holistic approach towards the family of diacylglycerol-binding C1 domain proteins and so includes PKCs and chimaerins. In collaboration with Marcelo Kazanietz (Univ. of Pennsylvania) we characterized the strucure of beta2-chimaerin. We used this information to elucidate the activation mechanism, and engineered a mutant that is supersensitive to DAG activation. This turbo-chimaerin mutant was then used to elucidate the physiological regulatory action of beta2-chimaerin in the Kazanietz lab.[unreadable] Structural and functional studies of C1 domains have established the outlines of a mechanism for diacylglycerol and phorbol ester-induced translocation to membranes. Concomitant with membrane translocation, PKCs, chimaerins, and other C1 domain proteins become allosterically activated. The PKC activation mechanism is thought to require a large conformational change, on the basis of changes in susceptibility to limited proteolysis, translocation kinetics, fluorescence, surface pressure analysis, and other indirect structural techniques.